The long-term goal of this project is to elucidate the cellular mechanisms of parathyroid hormone receptor (PTH1R) action. The PTH1R regulates extracellular calcium and phosphate homeostasis by its actions on kidney and bone. Like other G protein-coupled receptors, the PTH1R exhibits a cyclical process of activation, desensitization, internalization, and resensitization. Receptor desensitization provides a mechanism to protect cells against excessive stimulation, while resensitization guards cells against prolonged inactivity and hormone resistance. Unlike most other receptors, however, the PTH1R exhibits considerable cell- and tissue-specific differences in its activation. These differences cannot be ascribed to alternatively spliced receptor forms, receptor abundance, or G protein availability. Recent evidence suggests that the cytoplasmic adaptor protein ezrin-binding protein 50 kD (EBP50) may contribute to cell-specific PTH1R signaling and internalization. The central goal of the proposed studies is to examine the interaction and modulatory activity of EBP50 on all aspects of PTH1R cycling. Four specific aims are developed to test the unifying hypothesis that EBP50 regulates ligand-specific responses of the PTH1R. Aim 1 will characterize the effects of EBP50 on cell-specific PTH1R activation. Aim 2 will describe EBP50 effects on PTH1R desensitization. Aim 3 will identify structural determinants of EBP50 that are involved in PTH1R internalization. Aim 4 will determine the effects of EBP50 on PTH1R recycling. The planned studies employ an array of cell biological, biochemical, and molecular biological techniques that will be applied to specific kidney and bone cells that are the primary targets of PTH action. Preliminary data provide provisional support and establish the feasibility for much of the proposed work. The planned studies will yield novel and important information on the mechanism and role by which the PTH1R regulates extracellular calcium homeostasis. The results will provide greater understanding of the initiation and termination of PTH1R action. The outcomes may suggest additional pathophysiological mechanisms causing PTH resistance and lead to new treatment opportunities.